Glycyrrhetinic Acid Induces Apoptosis in Leukemic HL60 Cells Through Upregulating of CD95/ CD178.

Acute leukemia is characterized by the accumulation of neoplastic cells in the bone marrow and peripheral blood. Currently, chemotherapy and differentiating agents have been used for the treatment of leukemia. Recently, plant extracts, either alone or in combination with chemo agents, have been proposed to be used for the treatment of cancers. The aim of the present research was to study the cytotoxicity and apoptosis effects of an active licorice-derived compound, glycyrrhetinic acid (GA), on human leukemic HL60 cells. HL60 cells were cultured in RPMI1640 containing 10% fetal bovine serum. Cells were treated with different doses of GA and their viability and proliferation were detected by dye exclusion and 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays. Apoptosis induction and expression of CD95 and CD178 were analyzed by flow cytometry. We observed that GA decreases cell viability and suppresses cells proliferation in a dose- dependent manner. In addition, our flow cytometry data show that GA not only induces apoptosis in HL60 cells, but also upregulates both CD95 and CD178 expression on the cell surface of these cells in a dose-dependent manner. The combination of GA with cytotoxic drugs and differentiation agents requires further investigation.

Apoptosis or programmed cell death is one of the fundamental mechanisms that regulates cell growth and death (3). Basically, apoptosis is activated by CD95 which binds to its ligand, CD178 on the activating cell which is often a lymphocyte (4).
Interaction of CD95 and CD178 leads in activation of death domains which subsequently results in activation of caspase and cell death (4,5 valuable resources for production of pharmaceutical products and chemo drugs (7,8). Licorice is a traditional herbal medicine originated from the licorice root, and it remains one of the most commonly prescribed herbs in Chinese and Persian medicine (9,10). Glycyrrhetinic acid β (GA), the hydrolyzed metabolite of glycyrrhizin (GZ), exhibits many anti-inflammatory and anti-viral effects. Moreover, previous studies revealed that GA and GZ showed toxic effects against many cancer cells including liver, prostate and colon (11)(12)(13). However, its underlying mechanism still needs to be explored.
The aim of this study was to evaluate the cytotoxic effects of GA against the growth and proliferation of HL60 cells as a promyelocytic leukemia cell line and investigate its effects on CD95 and CD178 expression as activators of apoptosis.

Cell culture
The HL60 cell line (human promyelocytic leukemia cell line) was purchased from Pasteur Institute of Iran (Tehran, Iran) and was cultured in RPMI 1640 (Gibco, Manchester, UK) containing 10% FBS, at 37°C in a humid incubator with 5% CO2.

Cell viability
To examine the effect of GA on cell viability
Thus, XTT assay (CCK-8, Dojindo,Tokyo, Japan) was employed to assess the toxic effects of GA on

Statistical analyzes
Arithmetic means and standard deviations were calculated and statistical significance was defined as P ≤ 0.05 using Student's t-test.

GA inhibits cell viability and proliferation
First, we examined the effects of GA on the viability of HL60 cells and observed that this licorice-derived compound is capable to decrease cell viability in a dose-dependent manner ( Figure   1A). Moreover, the effect of GA on cell proliferation was evaluated using XTT assay. Our data show that GA inhibits cell proliferation in a dose-dependent manner ( Figure 1B (Figure 4 A). Interestingly, we observed that CD178 is not expressed on the cell surface of HL60 cells, but its expression enhanced when the cells were treated with GA ( Figure 4B).

Discussion
Currently, various therapeutic methods are being used to destroy malignant leukemic clones.  In contrast to our data, this previous report showed that GA was only toxic to gastric cell lines when treated with a high dose of GA (16). Similarly, another study has reported that GA is not capable to induce apoptosis in rat hepatocytes (17). These Mounting evidence indicates that many chemo drugs induce apoptosis in leukemic cells (2).
However, leukemic cells become resistant when they express multi-drug resistant molecules such as P-glycoprotein (18)   Having shown that GA upregulates expression of both CD95 and CD178 on the cell membrane of HL60 cells, we postulate that the interaction of elevated CD95 and CD178 could lead to the induction of apoptosis in GA-treated HL60 cells.
In conclusion, we herein demonstrated that GA exhibits cytotoxic activities against HL60 cells by induction of apoptosis and that GA-induced apoptosis is at least partially initiated by interaction of CD95 and CD178 signaling pathway.